Hemorrhagic fever viruses are of serious worldwide health concern as well as potential biological weapons. Lassa fever virus in particular annually infects several hundred thousand individuals in West Africa, and the export of this pathogen outside of this region, either intentionally or unintentionally, presents a serious risk to the developed countries of the world. The CDC and NIAID have identified Lassa fever virus as a Category A priority pathogen, indicating the highest degree of threat to public health. The purpose of our biodefense program is to develop safe and effective drugs for preventing and treating diseases caused by Category A viruses. To that end, a large and diverse library of small molecule compounds was screened to identify inhibitors that target the essential Lassa surface glycoprotein and thus block viral entry into the host cell. A number of these potent antiviral compounds and their related analogs have exhibited informative chemical structure-biological activity relationships (SAR). The goal of the Phase II proposal is to advance these compounds toward clinical development, to submit an application for an Investigational New Drug (IND) for the strongest candidate, and to identify at least one additional preclinical candidate as a potential back-up compound if needed. Lead compounds will be optimized by criteria of potency, toxicity, metabolism, and pharmacokinetics. Compounds that demonstrate acceptable traits will be evaluated for efficacy in animal models of Lassa fever. These experiments will be performed initially in guinea pigs, with efficacious compounds further tested in nonhuman primates. In addition, the mechanism of antiviral action will be explored both directly and by examination of prospective viral escape mutants. Replication-deficient retroviruses that incorporate a heterologous envelope protein such as the Lassa fever virus glycoprotein (viral pseudotypes) will be used to assess compound potency and mechanism of action; these experiments can be performed using biosafety level 2 (BSL-2) conditions. Results will be validated in BSL-4 facilities using authentic Lassa fever virus; animal studies using Lassa fever virus will also be conducted in BSL-4 facilities. [unreadable] [unreadable] [unreadable]